PROJECT SUMMARY/ABSTRACT Treatment of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) and pulmonary infections caused by non-tuberculous mycobacteria (NTM) is lengthy and complex, requiring patients to take multiple drugs for a minimum of 6 months. There is a critical need to define new targets for development of antibiotics to shorten and simplify treatment of TB and NTM infections. Most current antibiotics target essential functions. While genes essential for Mtb growth in standard in vitro conditions have been defined, these genes may not be required during infection. The overall goal of this proposal is to determine if specific pathways annotated as essential for in vitro growth are required for Mtb growth or persistence in the host. The proposed research will test the central hypothesis that only a subset of functions essential for in vitro growth will be essential during infection due to differences in the availability of metabolic precursors. Analyzing the importance of in vitro- essential genes to Mtb infection typically requires conditional expression methods. While these methods are valuable for individual analysis of gene essentiality they remain slow and inefficient, making them inappropriate for discovery. Specific Aim 1 will make use of a novel collection of transposon (Tn) insertion mutants in genes previously annotated as essential for in vitro growth generated using a custom rich medium. An innovative transposon sequencing (Tn-seq) approach will be taken using defined mini-libraries of these Tn mutants and a mouse high-dose aerosol infection model to rapidly and efficiently identify those in vitro-essential genes that Mtb requires for growth in the host. Preliminary data suggest that ribA2, encoding the first committed step in riboflavin biosynthesis, is one in vitro-essential gene required for Mtb infection. Mammals do not encode the riboflavin synthesis enzymes, making them promising drug targets. Specific Aim 2 will test the hypothesis that Mtb requires riboflavin biosynthesis for growth and persistence in the host using state-of-the-art conditional expression technologies and a mouse infection model. The proposed research is expected to identify several in vitro-essential genes that Mtb requires for infection and to demonstrate that de novo riboflavin biosynthesis is a function essential for both replication and persistence of Mtb in the host. This knowledge will be significant because it will define new targets for the development of antibiotics to treat TB and pulmonary NTM infections.